Characterization and evaluation of the therapeutic benefits of pure and lanthanides mono- and co-doped zinc oxide nanoparticles

The effect of Lanthanides-doping on the structural, optical, morphological, antibacterial and anticancer properties of zinc oxide (ZnO) nanoparticles was investigated. Pure ZnO, Zn0.9La0.1O, Zn0.9Ce0.1O, and Zn0.9La0.05Ce0.05O were fabricated through the chemical co-precipitation route. The structural and morphological properties were studied using the X-ray diffraction (XRD) and transmission electron microscopy (TEM), respectively. The optical properties were analyzed by photoluminescence spectroscopy (PL). The inhibitory effect of the synthesized nanoparticles (NPs) was assessed against six bacterial strains using the agar well diffusion and broth micro-dilution methods. The anticancer potential of the synthesized NPs was assessed against two human colon cancer cell lines Caco-2 and HCT-116. The appearance of the La2O3 and CeO2 secondary phases upon doping La3+ and Ce3+ ions induced structural and morphological changes. The large distorted hexagonal morphology of pure ZnO is transformed into small sized distorted hexagonal form. The photoluminescence spectra revealed the point defects resulting from Lanthanum (La) and cerium (Ce) doping. The prepared NPs significantly inhibited the growth of the six investigated bacteria and induced cytotoxic effects and morphological changes against Caco-2 and HCT-116 cell lines. This study showed that doping ZnO with lanthanide ions such as La3+ and Ce3+ provide promising biological applications. These NPs showed a potent antibacterial and anticancer effect towards the investigated bacterial strains and colon cancer cell lines. These findings point to the importance of the biological applications of NPs, and the possibility of investigating other biomedical applications for NPs.


Introduction
Nanotechnology is the study of manipulating and controlling matter on the nanoscale level utilizing logical insight into different biotechnological applications (Jeevanandam et al., 2018). A wide range of nanoscale materials with unique physical, chemical, optical and magnetic properties have recently been developed in this field (Khan et al., 2019). Considering these unique properties, NPs can be used in several biomedical, industrial, healthcare, and envi-ronmental applications (Khan et al., 2019). In particular, metal and metal oxide NPs have garnered considerable attention due to their highly potent antibacterial and anticancer effects (Dizaj et al., 2014;Hamida et al., 2020a;Vinardell and Mitjans, 2015). ZnO NPs exhibited the best bactericidal effect towards different bacterial strains and induced higher toxicity against various cancer cell lines than other metal and metal oxide NPs (Azam et al., 2012;Abbott Chalew and Schwab, 2013). In addition, ZnO NPs are of special relevance for their therapeutic benefits as antibacterial and anticancer agents (Jiang et al., 2018). Although ZnO NPs possess remarkable properties which make them suitable for various technological applications, these NPs showed limited stability in biological applications due to their instability in water (Lallo da Silva et al., 2019). Thus, doping can significantly enhance the physical, chemical, photocatalytic, antibacterial, and anticancer properties of ZnO Nps (Carofiglio et al., 2020;Lallo da Silva et al., 2019). In particular, doping with lanthanides is vital due to their distinctive features (Lee et al., 2017;Wen and Wang, 2014;Nabeel, 2020). In this context, La-doped ZnO nano-collides have exhibited substantial inhibiting activity against a set of bacteria and brine shrimps due to morphology changes, including shape modification and a decrease in the NPs' size (Shahzad et al., 2019). On the other hand, the high concentration of Ce present in Ce-doped ZnO samples possessed more inhibitory activity toward bacteria than the lower dopant concentration (Bomila et al., 2017). In addition, the simultaneous doping of La 3+ and Ce 3+ ions into the ZnO matrix induced a better inhibitory effect on a set of bacteria when compared with the simultaneous doping of either La 3+ or Ce 3+ ions with gadolinium ion (Gd 3+ ) (Bomila et al., 2019). Furthermore, lanthanide ions (La 3+ , Ce 3+ , and Neodymium (Nd 3+ ))-doped ZnO NPs exhibited various degrees of toxicity against A498 cells with minimal toxicity towards normal Vero cells and showed a potent inhibitory effect (Karthikeyan et al., 2019). Until now, the mechanism by which NPs inhibited bacteria and induced cytotoxic effects is still poorly understood. Different studies have proposed that the cellular interaction of NPs enhances the reactive oxygen species (ROS) production, which affects the structure of DNA, enzymes, and lipids and may result in cell damage (Hamida et al., 2020b(Hamida et al., , 2020cUllah et al., 2022). Many techniques have been used for the synthesis of lanthanides-doped ZnO NPs, such as coprecipitation (Theivarasu and Indumathi, 2017), sonochemical (Phuruangrat et al., 2016), gel combustion (Nguyen et al., 2019), and biological synthesis (Karthikeyan et al., 2018a). The coprecipitation method is used in the present work since it is a cost-effective method, in addition to its simplicity, better control of particle morphology and composition, and easy control of the pH and sintering temperature (Almoussawi et al., 2020). In this context, it was reported that doping ZnO with lanthanide ions, synthesized via the precipitation method, enhanced the antibacterial and anticancer potential compared with pure ZnO NPs (Manikandan et al., 2017;Nabeel, 2020;Theivarasu and Indumathi, 2017). Based on the literature, few studies have reported the biological activity of lanthanides mono-and codoped ZnO NPs. On the other hand, capping agents can significantly influence the morphology and stability of the NPs throughout the synthesis process (Chandrasekaran et al., 2012;Javed et al., 2016). Ethylenediamine tetra acetic acid (EDTA) is particularly relevant since it has high capping suitability on the surface of NPs, allowing the NPs to be highly water-dispersible, and can be used as a stabilizer (Jang et al., 2018;Nithiananth and Velraj, 2016;Yi et al., 2014). Knowing that EDTA comprises six reaction sites, four of which are hydroxyl groups that can form coordination bonds with Zn 2+ ions and so efficiently affect the morphology of the NPs during crystal formation (Chandrasekaran et al., 2012).
The purpose of this work is to evaluate the biological activity of pure ZnO, Zn 0.9 La 0.1 O, Zn 0.9 Ce 0.1 O, and Zn 0.9 La 0.05 Ce 0.05 O synthesized using the chemical co-precipitation technique and capped with EDTA. The characterization of the synthesized NPs was studied using the XRD, TEM, and PL analysis. The antibacterial potential of the synthesized NPs was investigated towards six bacterial strains including Escherichia coli, Citrobacter braakii, Klebsiella pneumonia, Staphylococcus aureus, Streptococcus intermedius, and Staphylococcus haemolyticus. In addition, the antitumor effect of these NPs was assessed towards two human colon cancer cell lines (Caco-2 and HCT-116).

Fabrication process
The chemical co-precipitation process was used for the fabrication of pure ZnO (Al Bitar et al., 2022), Zn 0.9 La 0.1 O, Zn 0.9 Ce 0.1 O, and Zn 0.9 La 0.05 Ce 0.05 O. An adequate amount of ZnCl 2 , LaCl 3 , CeCl 3 Á7H 2 O, and 0.1 M EDTA were weighed and then dissolved in distilled water as a dispersing solvent. The solutions were then titrated by 4 M NaOH, added in drop-wise with constant stirring, until pH reached 12. The solutions were then heated for 2 h at 60 ℃ with constant stirring, and washed several times with distilled water until the pH reduced to 7. The obtained white precipitates were then dried at 100 ℃ for 18 h, ground and calcinated at 550 ℃ for 4 h. From now on, pure ZnO, Zn 0.9 La 0.1 O, Zn 0.9 Ce 0.1 O, and Zn 0.9 -La 0.05 Ce 0.05 O will be referred to as ZnO-Pure, ZnO-La, ZnO-Ce, and ZnO-LaCe, respectively.
The prepared samples were characterized by X-ray Diffraction (XRD) using Bruker D8 Focus powder diffractometer, with CuK a radiation (k = 1.54056 Å) in the range of 25° 2h 80°. The surface morphology of the prepared NPs was measured using Transmission electron microscope (TEM) JEM-1400 Plus. Using the photoluminescence spectrophotometer FP-8600 with an excitation wavelength of 325 nm and a wavelength ranges from 350 to 700 nm, the emission spectra of the synthesized NPs were examined.

Cell culture
Two human colon cancer cell lines Caco-2 and HCT-116 were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Cells at the 8-12 passages were used in the experiments. The cell lines were cultured in DMEM culture medium supplemented with 10 % FBS, 1 % Penicillin-Streptomycin antibiotic (biowest, France), and 1 % L-Glutamine (Sigma-Aldrich, Germany). The cells were incubated in a humidified atmosphere (5 % CO 2 and 37 ℃).

MTT assay
100 ll of culture medium containing 5 Â 10 5 =ml of HCT-116 or Caco-2 cells were seeded to each well of 96-well plate. DMEM culture medium was used as the vehicle for the prepared NPs, and six replicates were considered for each concentration (0.1-3.2 mM). Wells having culture medium only were considered as a negative control. Cells were incubated for 24 h in order for the cells to adhere to the plate bottom (5 % CO 2 and 37 ℃). Cells were incubated with increasing concentrations of the NPs for 24-48 h. Following the incubation period, 10 ll of 5 mg/ml MTT solution was added to the cells, and the cells were incubated for 3-4 h. After labeling the cells with MTT, as described above, the solution was removed and 50 ll of isopropyl alcohol solution was added to each well and mixed thoroughly, then incubated for additional 10 min at 37°C. Subsequently, the plates' light absorption was measured at 540 nm in a microplate (ELISA) reader.
2.5. Cell morphological analysis and crystal violet staining 5 Â 10 5 /ml HCT-116 or Caco-2 cells were seeded in 12-well plates, cultured in DMEM complete media, and then incubated for 24 h (5 % CO 2 and 37 ℃). Cells were incubated with increasing concentrations of the NPs (0.1-1.6 mM) for 24-48 h. The morphological changes were visualized, examined, and images were captured using Leica DM500 microscope and compared with control untreated cells.
2.6. Antibacterial activity of the NPs 2.6.1. Microbial strains and antibacterial activity of the synthesized NPs using the agar well diffusion method Six bacteria were used for the assessment of the antibacterial activity of the prepared NPs. These bacteria included gramnegative (Escherichia coli, Citrobacter braakii, and Klebsiella pneumonia) and gram-positive (Staphylococcus aureus, Streptococcus intermedius, and Staphylococcus haemolyticus) bacteria. Escherichia coli, Citrobacter braakii, Staphylococcus aureus, Staphylococcus haemolyticus, and Streptococcus intermedius were isolated from the waste water collected from a station related to South Lebanon Water Establishment (SLWE) in Sidon city and Klebsiella pneumonia obtained from Mount Lebanon Hospital (UMC). The identification of the isolated bacteria was made using the VITEK analysis. The same experimental procedure was done as discussed and illustrated by Al Bitar et al. (2022) to assess the inhibitory effect of the synthesized NPs (200, 100, 50, and 25 mg/ml) against the six investigated bacteria. Serial half-fold dilutions of the three antibiotics (AB; Ciprofloxacin, Amoxicillin, and Doxycycline) (12.5 mg/ ml, 6.25, 3.125, and 1.5625 mg/ml) and sterile distilled water were used. The zone of inhibition (ZOI) was measured and presented as the mean AE the standard error of the mean (SEM) after incubating the plates for 24 h at 37 ℃. Methicillin Resistance test was done on the two staphylococcus species (Staphylococcus haemolyticus and Staphylococcus aureus). Resistance to Cefoxitin (30 lg) indicated that the tested Staphylococcus species are Methicillin Resistance ( Figure S1).

Minimum inhibitory concentration (MIC)
In order to quantify the antibacterial activity, the MIC values of the prepared NPs were estimated using the broth micro-dilution method using 96-well U-bottom microtiter plates. Bacterial suspension of the six bacteria was done by transferring the fresh colonies grown overnight on NA plates to sterile test tubes containing nutrient broth. Serial half-fold dilution of the NPs was prepared by dispersing the NPs in nutrient broth. The test was carried out by distributing 100 ll per well bacterial suspension with final inoculum concentrations of 10 6 CFU/ml (Iseppi et al., 2020). Then, this 100 ll per well bacterial suspension was treated with 100 ll of the prepared NPs dilutions (triplicate) to achieve the desired concentrations ranging between 200 and 0.01221 mg/ml. After 24 h, the MIC was determined by measuring the optical density (OD) at 590 nm. The OD values for the wells with concentrations ranging between 12.5 and 0.01221 mg/ml were registered and plotted.

Minimum bactericidal concentration (MBC)
The MBC was determined after broth micro-dilution by subculturing the different concentrations ranging between 200 mg/ ml À 0.01221 mg/ml from wells on MHA plates. The plates were then incubated for 24 h at 37°C and observed for bacterial growth (Jam et al., 2022).

Statistical analysis
The obtained data were represented as the mean AE SEM of at least three independent replicates, and two-way ANOVA was used for the statistical analysis using the GraphPad Prism 6 program (Ver. 6.01). p values were calculated such that p > 0.05 was not significant (ns), *p < 0.05, ** p < 0.01, and *** p < 0.001, where *, **, and *** denote levels of significance.

Structural and optical analysis
To examine the purity of the ZnO phase, the Maud program was used for the structural Rietveld refinements of the X-ray diffractogram patterns of ZnO-Pure (Al Bitar et al., 2022), ZnO-La, ZnO-Ce, and ZnO-LaCe (Fig. 1). These patterns are indexed to the ZnO wurtzite structure with space group P6 3 mc and elven main diffraction peaks, corresponding to the crystalline planes (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3), (2 0 0), (1 1 2), (2 0 1), (0 0 4), and (2 0 2), respectively (AL-Asady et al., 2020; Almoussawi et al., 2020;Kumar and Sahare, 2014;Morkoç and Özgür, 2009). In addition, the detection of secondary peaks in the obtained patterns is due to the doping with La 3+ and/or Ce 3+ ions. These secondary peaks correspond to the appearance of secondary phases as La 2 O 3 in the ZnO-La sample, CeO 2 in the ZnO-Ce sample, and both La 2 O 3 and CeO 2 phases in the ZnO-LaCe sample (Fig. 1). The percentages of the secondary phases present in the synthesized NPs, and the refined lattice parameters a and c values with their corresponding ratios c=a ð Þobtained from the Maud program are listed in Table 1. The values of the refined lattice parameters show a slight change upon doping. ZnO-La and ZnO-LaCe samples exhibit lower a values, whereas the ZnO-Ce sample exhibits higher a value compared to the ZnO-pure sample. Furthermore, the three doped samples register lower c values compared to the ZnO-pure sample. In order to determine the deviation of the crystal lattice from the perfect arrangement, the degree of distortion (R) is calculated using equation (1) (Yasmeen et al., 2020). The obtained c=a ð Þ ratios and the degree of distortion (R) stay almost constant around (1.60) and (1.019), respectively.
On the other hand, the average crystallite size (D DSM ) values are calculated using the Debye -Scherrer model (DSM), given by equation (2) (Farhat et al., 2018): where k, k, b hkl and h represent the shape factor constant (0.9), CuK a X-ray wavelength, full width at half maximum and peak position, respectively. D DSM values are computed from the plot's slope, fitted according to equation (2). The obtained average crystallite size decreases upon doping with La 3+ and/or Ce 3+ ions (Table 1). Non-agglomerated NPs are seen in the TEM images with different average sizes and distorted hexagonal shapes (Fig. 2). The average particle size (D TEM ) of the synthesized NPs is obtained from the particle size distribution histogram, as shown in Fig. 2(a). ZnO-Pure NPs demonstrate a distorted hexagonal shape having an average particle size of 70.614 nm (Al Bitar et al., 2022). The influence of doping with La 3+ and/or Ce 3+ ions on ZnO NPs evident, as shown in Figures (2b, c, and d). The average particle size decreases to 29.43 nm with La doping, whereas it decreases more to 7.23 nm with Ce doping (Table 1). However, the simultaneous doping of La 3+ and Ce 3+ ions in the ZnO-LaCe sample shows two distinguish-able distorted hexagonal shapes with two average particle sizes of 72.44 and 10.56 nm. The D TEM values obtained from the particle size distribution histograms showed insignificant variation compared to the D DSM values calculated using the Debye -Scherrer model (DSM), as shown in Table 1.
The PL spectra of ZnO-Pure (Al Bitar et al., 2022), ZnO-La, ZnO-Ce, and ZnO-LaCe samples were examined in the range of 350-700 nm, as displayed in Fig. 3. All the samples demonstrate two UV emission peaks corresponding to the near band edge emission (NBE). Peak located at 387 nm is shifted to a lower wavelength value with La or Ce doping in ZnO-La and ZnO-Ce samples, whereas it is shifted to a higher wavelength value with the simultaneous doping of La 3+ and Ce 3+ ions in the ZnO-LaCe sample. In the UV region, the ZnO-LaCe sample registers the lowest intensity compared to other samples (Fig. 3). However, La doping results in a remarkable increase in the intensity of the NBE peaks, which means La 3+ ions induce more excitons (Obeid et al., 2019). Furthermore, the emission peaks corresponding to deep-level (DL) emis-sion can be attributed to several structural defects. These include oxygen or zinc vacancies, oxygen or zinc interstitial, or oxygen antisites present in the samples (Mishra et al., 2010). Accordingly, the intensity of the peaks due to DL emission in the visible region decreased upon incorporating La 3+ and/or Ce 3+ ions (Fig. 3).

Antibacterial activity
The inhibitory effect of ZnO-Pure (Al Bitar et al., 2022), ZnO-La, ZnO-Ce, and ZnO-LaCe samples was tested against six bacterial strains (Escherichia coli, Citrobacter braakii, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus intermedius, and Staphylococcus haemolyticus) using the agar well diffusion and broth microdilution methods. The calculated ZOI results of the synthesized NPs with the standard antibiotics (Ciprofloxacin, Doxycycline, and Amoxicillin) are presented in Fig. 4 and listed in Table S1. The existence of an inhibition zone demonstrates the inhibitory effect of the standard antibiotics and the prepared samples (Fig-Fig. 1. X-ray diffractogram patterns with their corresponding Rietveld refinements of ZnO-Pure, ZnO-La, ZnO-Ce and ZnO-LaCe NPs performed on the MAUD software. Note: *, Secondary peaks. ures S2, S3, and S4). The confidence interval, p values, and the significance level are listed in Tables S2, S3, and S4. As the concentration of the three used antibiotics increases, the inhibitory effect increases (Fig. 4). Among the used antibiotics, Ciprofloxacin registered the highest antibacterial activity against the investigated bacteria. Escherichia coli, Citrobacter braakii, and Klebsiella pneumoniae were the most bacteria sensitive to ZnO-Pure NPs, as reported previously by Al Bitar et al. (2022) Table 2. Escherichia coli was highly sensitive to ZnO-Pure NPs with an MIC value of 1.563 mg/ml, compared to ZnO-La and ZnO-Ce, with an MIC of 3.125 mg/ml (Table 2). On the other hand, Citrobacter braakii was the most bacterium responsive to ZnO-Pure NPs with an MIC value of 1.563 mg/ml, whereas it showed a higher MIC value of 6.250 mg/ml for ZnO-Ce and ZnO-LaCe samples (Table 2). However, Klebsiella pneumonia was found to be most susceptible to the ZnO-La sample and registered the lowest MIC value of 0.391 mg/ml compared to ZnO-Pure and ZnO-Ce samples with 3.125 and 6.250 mg/ml, respectively (Table 2). In contrast, Staphylococcus aureus and Staphylococcus haemolyticus were highly responsive to the simultaneous doping of La 3+ and Ce 3+ ions with low MIC values of 0.391 and 0.781 mg/ml, respectively (Table 2). In addition, in the present study, all the samples showed no minimum bactericidal concentration (MBC).

Anticancer activity
The anticancer potential of the synthesized samples was evaluated against Caco-2 and HCT-116 cell lines using the MTT assay. The IC 50 values were calculated after treating the cells with increasing concentrations of the NPs (0.1-3.2 mM) for 24-48 h. At 24 h, ZnO-LaCe and ZnO-La samples showed the highest toxicity against Caco-2 and HCT-116 cells compared to the other prepared samples with IC 50 values 0.93 and 0.48 mM, respectively. At 48 h, the calculated IC 50 value against Caco-2 cell line after exposing the cells to ZnO-Pure treatment was 0.78 mM; however, doping ZnO with La 3+ and/or Ce 3+ ions induced less toxicity against this investigated cell line and showed higher IC 50 values, such that the calculated IC 50 values for ZnO-La, ZnO-Ce, and ZnO-LaCe samples were 1.58, 0.94, and 0.91 mM, respectively (Fig. 5). Furthermore, doping with Ce enhanced the cytotoxic effect of ZnO-Pure NPs against HCT-116 cell line (IC 50 = 0.52 mM) for 48 h. The obtained data showed that the synthesized NPs induced a significant decrease in the proliferation of the cells in a dose-and time-dependent manner compared to the untreated control cells at concentrations above 0.4 mM ( Figure S7).
The cell morphological changes were examined for both treated cell lines with the synthesized NPs concentrations (0.1-1.6 mM) after 24 and 48 h (Fig. 6, Figures S8, S9, S10, and S11). No significant difference in morphology was seen between the control and the treated cells at low concentrations (0.1 and 0.2 mM). However, at higher concentrations (0.4, 0.8, and 1.6 mM), the treated cells showed remarkable alteration in normal morphology, cell shrinkage, and size reduction. The reduction of the viable cells was observed in the treated cells by staining the cells with crystal violet solution ( Figure S12). This verified the MTT assay results of cell proliferation and cytotoxicity induced by the synthesized NPs.

Discussion
The obtained X-ray diffractogram patterns with their corresponding Rietveld refinements reveal the fingerprint of the hexagonal wurtzite structure (Fig. 1). The formation of the secondary phases in the three doped samples can be linked to the larger ionic radii of La 3+ and Ce 3+ ions compared to Zn 2+ ions, preventing the substitutional incorporation of La and/or Ce in Zn-sites and causing deposition on the ZnO surface (Ahmad et al., 2015;Bomila et al., 2019;Suwanboon et al., 2013). The change in the a and c values may be due to the formation of the secondary phases or the mismatch of the ionic radii of the dopants La 3+ and Ce 3+ ions and host Zn 2+ ions (Iqbal et al., 2009;Suwanboon et al., 2013). The calcu-lated R values are greater than unity (R > 1), indicating crystal lattice distortion (Yasmeen et al., 2020). The constant values of c=a ratios and R indicate that doping with La 3+ and/or Ce 3+ ions does not cause a considerable impact on the wurtzite hexagonal structure of the ZnO host lattice (Bomila et al., 2017;Labhane et al., 2018). The particle size and degree of crystallinity of the three doped samples decreased, as indicated by the XRD peaks broadening with the decrease in their intensities (Pal et al., 2012;Poornaprakash et al., 2016). A Similar phenomenon was observed by Ahmad et al. (2015) in the case of doping ZnO NPs with Ce ions. Ahmad et al. (2015) suggested that the localization of the dopant ions in or near the boundary of ZnO NPs may reduce the diffusion rate and hinder the NP growth, resulting in a decrease in their crystallite size. The morphology ''size and shape" of ZnO-Pure NPs was modified via doping with La 3+ and/or Ce 3+ ions and using EDTA as a capping agent (Fig. 2). As reported in the literature, the NP morphology of doped ZnO with La 3+ and/or Ce 3+ ions showed either a spherical or rod shape (Bomila et al., 2019;Goel et al., 2017;Jayachandraiah et al., 2014). Similarly, it was reported that other lanthanides (Ln 3+ = Eu 3+ , Gd 3+ , Sm 3+ , Pr 3+ , and Er 3+ )-doped ZnO-NPs, fabricated without using a capping agent, exhibited nanorod, irregular, and non-uniform morphologies (Chen et al., 2018;Farhat et al., 2018;Nabeel, 2020). However, in the present study, the NP shape prepared via the co-precipitation method was controlled using EDTA as a capping agent, and a distorted hexagonal form was obtained. The estimated values of D TEM are different from the D DSM values obtained using the XRD studies, as displayed in Table 1. ZnO-Pure and ZnO-La NPs registered higher D TEM values compared to the estimated D DSM values. For D TEM greater than D DSM , this difference can be attributed to the accumulation of crystals in the TEM technique due to grain growth, as reported by Kamareddine et al. (2020). As a result, particles composed of several crystallites are formed, each corresponding to a coherent diffraction domain. However, it can be assigned to the reaction solution aging in the case of D TEM smaller than D DSM , as reported by Bitar et al. (2017). In the present case, the particle size is highly affected by the aging in the reaction solution, particularly in ZnO-Ce and ZnO-LaCe samples, due to the doping with Ce 3+ ions.
The PL spectra of all the synthesized samples exhibited both UV and DL emission peaks (Fig. 3). The UV emission peaks are due to the recombination of electron-hole pairs corresponding to the NBE transition of ZnO NPs (Lang et al., 2010;Sharma and Jha, 2017). It was reported that the strain induced in the crystal lattice via doping might be responsible for the shift in the UV emission peak (Pandey et al., 2015). The decrease in the PL intensity in the UV region could imply a lower electron-hole recombination rate, leading to higher photocatalytic activity (Flores-Carrasco et al., 2021;Lang et al., 2016). In addition, the quenching of the peaks due to DL emission in the visible region may be caused by photo-generated electrons being trapped in the trap centers (Limaye et al., 2011;Patel et al., 2017). It was reported that the visible color emissions could be useful for cell labeling applications (Tang et al., 2010). Moreover, the presence of defects and oxygen vacancies play a crucial role in enhancing the antibacterial effects (Bomila et al., 2017).
The synthesized samples demonstrated significant antibacterial activity on the investigated bacteria (Fig. 4). ZnO-Pure NPs inhibited the growth of the three tested gram-negative bacteria and staphylococcus aureus. In comparison with the three doped samples, ZnO-Pure NPs have shown the highest antibacterial activity towards Escherichia coli, Citrobacter braakii, and Klebsiella pneumonia and showed a small inhibitory effect against Staphylococcus aureus. Doping ZnO with either La 3+ or Ce 3+ ions caused a small suppression of Escherichia coli and Klebsiella pneumonia growth compared to ZnO-Pure NPs. However, doping with both dopant ions did not exert antibacterial activity toward these bacte-  slightly following treatment with ZnO-Ce and ZnO-LaCe NPs due to the presence of Ce 3+ ions. Only doping with La 3+ ions prevented the inhibition of this bacterium. Even though the tested Staphylococcus aureus was methicillin-resistant, all the prepared NPs fabricated using the co-precipitation method inhibited its growth. In this context, two studies were reported by Bomila et al. (2017Bomila et al. ( , 2019 investigating the antibacterial effect of mono-and co-doped ZnO NPs with rare-earth ions fabricated using the wet chemical method. In the first study, ZnO-Pure and ZnO-Ce samples showed no inhibitory effect towards Staphylococcus aureus at low concentrations and small inhibition against this bacterium at high concentrations (Bomila et al., 2017). In the second study, ZnO-Pure and the rare earth-doped ZnO NPs, including ZnO-LaCe samples, did not register an antibacterial effect against the tested Staphylococcus aureus (Bomila et al., 2019). Similarly, in the three studies conducted by Karthikeyan et al. (2018aKarthikeyan et al. ( , 2018bKarthikeyan et al. ( , 2019 the tested rare earth mono-doped ZnO NPs, fabricated using the green method, registered a minimal inhibitory effect against Staphylococcus aureus. Thus, the co-precipitation method used in this study is more beneficial than the fabrication methods mentioned in the literature. On the other hand, the growth of Staphylococcus haemolyticus was significantly inhibited only after treatment with ZnO-Ce and ZnO-LaCe NPs. In this study, among the three doped samples, ZnO-Ce and ZnO-LaCe NPs registered the highest antibacterial activity against Staphylococcus aureus and Staphylococcus haemolyticus, respectively. On the other hand, Streptococcus intermedius was the most bacterium that resisted the inhibitory action of ZnO-Pure, ZnO-La, and ZnO-Ce samples. Our results revealed that the simultaneous doping of La 3+ and Ce 3+ ions improved the antibacterial activity against this bacterium. Similarly, it was reported by Karthikeyan et al. (2018a) that the inhibition of bacterial growth was achieved by the ZnO-LaCe sample against the investigated bacteria; however, gram-positive bacteria were more responsive to the effect of doping. So far, the precise inhibitory action of NPs toward bacterial cells remains unclear. Some studies have shown that doping with La 3+ and/or Ce 3+ ions enhanced the inhibitory effect of ZnO NPs towards a set of bacterial strains (Karthikeyan et al., 2018a(Karthikeyan et al., , 2018bManikandan et al., 2017;Theivarasu and Indumathi, 2017). The authors attributed this enhancement activity to different factors, including the increase in dopants concentration, decrease in the particle size, and the release of La 3+ , Ce 3+ , and Zn 2+ ions. In our study, the variation in the antibacterial activity could be attributed to the difference in the morphology of the synthesized NPs, as shown in the TEM images (Fig. 2). This behavior could also be attributed to the appearance of the secondary phases obtained in the X-ray diffractogram patterns and verified by the Maud program ( Fig. 1) In addition, the interaction of the synthesized NPs with the bacterial cell wall may be impacted by the presence of defects and oxygen vacancies (Fig. 3). Based on the broth micro-dilution and agar well diffusion methods, the ZnO-Pure sample was highly responsive to Escherichia coli and Citrobacter braakii. However, the MIC value of the ZnO-La sample against Klebsiella pneumonia was found to be lower than those of the ZnO-Pure and ZnO-Ce samples. Furthermore, the MIC values registered by the ZnO-LaCe sample toward the two Staphylococcus species were lower than those of the rest investigated samples. Even though ZnO-Ce NPs registered higher MIC values when compared to ZnO-Pure, ZnO-La, and ZnO-LaCe NPs against the investigated bacteria, these MIC values did not exceed 6.250 mg/ml. In contrast, the MIC of Ce-doped ZnO NPs observed by Fifere et al. (2021) against both Staphylococcus aureus and Escherichia coli was 20 mg/ml, which was higher than that obtained by the present study. Accordingly, the synthesized samples inhibited the bacterial growth without inducing bacterial cell death. Thus, in the present study, the samples had a bacteriostatic effect only.
In the current study, Caco-2 and HCT-116 cell lines were chosen to evaluate the cytotoxicity effect of the synthesized NPs. Herein, the cytotoxic effect was examined by measuring the cell viability (%) of the treated cells with increasing concentrations of the NPs (0.1-3.2 mM) using the MTT assay for 24-48 h (Fig. 5). Various degrees of toxicity were induced by the prepared samples against the two human colon cell lines. The estimated IC 50 values were obtained in the range of 0.48-1.58 mM. Based on the obtained IC 50 values, ZnO-Pure and mono-doped ZnO NPs with either La 3+ or Ce 3+ ions exhibited more cytotoxicity towards HCT-116 cells than Caco-2 cells for both tested periods. In contrast, the obtained data revealed that substituting Zn 2+ ions with La 3+ and Ce 3+ ions exerted nearly the same cytotoxic effect toward both examined cells. The sensitivity of Caco-2 cells towards ZnO-LaCe and ZnO-Pure treatments for 24 and 48 h, respectively, was higher than that of HCT-116 cells. However, HCT-116 cells were more responsive to ZnO-La and ZnO-Ce treatments for 24 and 48 h, respectively. In addition, the treated cells with concentrations (! 0.4 mM) showed alterations in normal morphology and cell adhesion capacity compared to control cells (Fig. 6). For instance, Jasim and Saleh (2019) reported that ZnO NPs induced cytotoxicity and morphological changes in HCT-116 cells in a dose-dependent manner. Majeed et al. (2019) showed that bio-synthesized ZnO NPs induced significant cytotoxicity against HCT-116 cells with relatively less toxicity toward Vero cells. Similarly, El-Belely et al. (2021) verified that ZnO NPs reduced the proliferation of Caco-2 cells, whereas less toxicity was induced against normal lung cells. Furthermore, a recent study conducted by Aljohar et al. (2022) showed that chemically synthesized ZnO NPs exerted significant cytotoxic effect on human A431 skin carcinoma cells and displayed much lower toxicity to normal kidney Vero cells. This could be explained by the higher metabolic rates of cancer cells and thus their increased demand for nutrient acquisition, rendering them more sensitive than normal cells when exposed to the same treatments (Hammoudi et al., 2011;Ren et al., 2022). Sanaeimehr et al. (2018) showed that ZnO-pure NPs, synthesized using the green method, caused in killing 50 % of human liver cancer cells (HepG2) at concentrations greater than 87 lg/ml at 150 lg/ml after treating the cells for 24, 48, and 72 h. Similarly, Boroumand Moghaddam et al. (2017) reported that the green-synthesized ZnO-NPs induced an anticancer activity against breast cancer cells (MCF-7) with IC 50 equal to 121 lg/ml after exposing the cells for 24 h. In addition, Dulta et al. (2021) reported that green-synthesized ZnO-Pure NPs induced cytotoxicity in human cervical cancer cells (HeLa) and human colon cancer cells (HT-29) after 24 h with IC 50 values of 101.7 lg/mL and 124.3 lg/ml, respectively. On the other hand, Theivarasu and Indumathi. (2017) found that at 280 ± 0.05 lg/m l, chemically synthesized ZnO-Pure NPs using the coprecipitation method killed 50 % of (A549) human lung cancer cells. Compared with this mentioned literature, ZnO-Pure NPs fabricated in the present study showed a potent anticancer activity towards Caco-2 and HCT-116 cells with low IC 50 values ranging between 0.55 and 1.04 mM equivalent to 44.76 -84.64 lg/ml. However, the substitution of Zn 2+ ions by La 3+ and/or Ce 3+ ions enhanced the cytotoxic effect of ZnO NPs due to different factors, including the concentration and morphology of the NPs and level of ROS generation (Karthikeyan et al., 2019;Theivarasu and Indumathi, 2017). Shakir et al. (2016) showed that La-doped ZnO NPs exerted higher toxicity against different cancer cell lines than the pure sample treatment. Similarly, it was reported that Cedoped ZnO NPs, fabricated via the co-precipitation method, induced a more cytotoxic effect against (A549) human lung cancer with lower IC 50 values compared to the pure sample (Theivarasu and Indumathi, 2017). The authors attributed that to the increase of induced ROS production, resulting in apoptosis and cell death.
The current results showed that ZnO-Ce NPs induced a more cytotoxic effect towards HCT-116 cells with low IC 50 values and almost the same cytotoxic effect towards Caco-2 cells compared with the results obtained by Theivarasu and Indumathi. (2017). This enhancement in the anticancer potential of ZnO-Ce NPs could be attributed to the decrease in the particle size controlled using EDTA in the current study and the treated cell line. Based on the obtained results, EDTA significantly controlled the morphology of the prepared NPs, assured their stability, and enhanced their solubility in water, thus improving their biological efficiency.

Conclusion
The chemical co-precipitation technique allowed the successful synthesis of pure and lanthanides mono-and co-doped ZnO NPs with La 3+ and/or Ce 3+ ions, using EDTA as a capping agent. The wurtzite structure of ZnO NPs was validated by the XRD measurements and assured by the Reitveld refinements using the Maud program. Minor secondary phases of La 2 O 3 and/or CeO 2 were detected by the Maud program in the three doped patterns. Moreover, the morphology of ZnO NPs was greatly affected by the incorporation of La and/or Ce into the ZnO lattice and EDTA. The average particle size was reduced to a small-sized distorted hexagonal form with the appearance of two particle size distributions in the ZnO-LaCe sample. PL studies show the existence of several DL defects in the prepared NPs. This study showed that the prepared NPs exerted a significant cytotoxic effect on the two investigated human colon cancer cell lines and potent antibacterial efficiency toward the investigated bacteria. The IC 50 values range between 0.48 and 1.58 mM. The current data showed that ZnO-Pure, ZnO-La, and ZnO-Ce samples induced more toxicity against HCT-116 cells than the Caco-2 cells. However, the ZnO-LaCe sample induced almost the same cytotoxic effect on both investigated cell lines. The ZnO-Pure sample showed the highest antibacterial activity towards the gram-negative bacteria among the prepared samples. Only the ZnO-LaCe sample showed an inhibitory effect against Streptococcus intermedius. The three doped samples enhanced the antibacterial effect of ZnO NPs toward Staphylococcus aureus, whereas only ZnO-Ce and ZnO-LaCe samples showed an inhibitory effect toward Staphylococcus haemolyticus. The present study showed that lanthanides mono-and co-doped ZnO NPs might have the potential as an auspicious antibacterial and anticancer agent. Future investigations are needed to assess the in vivo toxicity effect and to illustrate more about the mechanism of action of the fabricated NPs.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.